Establishment of tolerance to commensal bacteria requires a complex microbiota and is accompanied by decreased intestinal chemokine expression

L. N. Fink, S. B. Metzdorff, L. H. Zeuthen, C. Nellemann, M. B. Kristensen, T. R. Licht, H. Frøkiær
2012 American Journal of Physiology - Gastrointestinal and Liver Physiology  
21 Intricate regulation of tolerance to the intestinal commensal microbiota acquired at birth is critical. 22 We hypothesized that epithelial cell tolerance towards early Gram-positive and Gram-negative 23 colonizing bacteria is established immediately after birth as has previously been shown for 24 endotoxin. Gene expression in the intestine of mouse pups born to dams, which were colonized 25 with either a conventional microbiota, L. acidophilus, E. coli or germfree, was examined on Day 1 26
more » ... mined on Day 1 26 and Day 6 after birth. Intestinal epithelial cells from all groups of pups were stimulated ex vivo with 27 L. acidophilus and E. coli to assess tolerance establishment. Intestine from pups exposed to a 28 conventional microbiota displayed lower expression of Ccl2, Ccl3, Cxcl1, Cxcl2 and Tslp than 29 germfree mice, whereas genes encoding proteins in TLR signaling pathways and cytokines were 30 up-regulated. When comparing pups on Day 1 and Day 6 after birth, a specific change in gene 31 expression pattern was evident in all groups of mice. Tolerance to ex vivo stimulation with E. coli 32 was only established in conventional animals. Colonization of the intestine was reflected in the 33 spleen displaying down-regulation of Cxcl2 when compared to germfree animals on Day 1 after 34 birth. Colonization reduced the expression of genes involved in antigen presentation in the 35 intestine-draining mesenteric lymph nodes, but not in the popliteal lymph nodes, as evidenced by 36 gene expression on day 23 after birth. We propose that microbial detection systems in the intestine 37 are upregulated by colonization with a diverse microbiota, while expression of pro-inflammatory 38 chemokines is reduced to avoid excess recruitment of immune cells to the maturing intestine. 39 40 42 43 the bacterial microbiota of the gut and communicate with the immune system via secretion of 61 cytokines and chemokines. As IEC chemokines recruit myeloid and lymphoid cells to the gut, they 62 are crucial in directing the early maturation of both the local and systemic immune system (9). 63 IECs exposed to microbe-associated molecular patterns become insensitive to further bacterial 64 stimuli (14; 32), indicating a critical mechanism in maintaining non-inflammatory conditions in the 65 gut. Part of the response of epithelial cell lines to TLR2 and TLR4 ligands is transfer of TLRs from 66 the apical to the cytosolic compartment, causing tolerance to subsequent ligand challenges (2; 23). 67 from the Danish Council for Animal Experimentation (Dyreforsøgstilsynet). 113 114 Preparation of bacterial suspensions 115 L. acidophilus NCFM was grown anaerobically in de Man, Rogosa, and Sharpe broth (MRS, 116 Merck, Darmstadt, Germany) and E. coli Nissle aerobically in Luria-Bertani broth (LB, Merck) 117 overnight at 37°C. The cultures were harvested, washed 2 times in sterile phosphate-buffered saline 118 (PBS, Lonza, Basel Switzerland), resuspended in PBS and frozen at -80 °C. For use in ex vivo 119 experiments, bacteria were killed by a 40 min. UV exposure prior to freezing. The endotoxin 120 concentration in L. acidophilus NCFM preparations were < 0.10 EU/ml measured by limulus 121 amoebocyte assay (Ass. of Cape Cod, East Falmouth, MA, USA). 122 123 Isolation of epithelial cells for ex vivo stimulation 124 At PND6, epithelial cells were isolated for ex vivo stimulation studies from small intestines of 2-3 125 pups per litter. The small intestines were placed in Hanks buffered saline (HBSS, Lonza), opened 126 longitudinally and cut in small pieces. The epithelial cells were detached from the underlying tissue 127 by incubation in fresh HBSS containing 2 mM EDTA at 37°C for 10 min. Residual tissue was 128 removed using a 70 μm filter. Cells were washed in cold PBS and resuspended in culture medium 129 (RPMI 1640 supplemented with 100 U/ml penicillin, 100 μg/ml streptomycin, 2 mM L-glutamine, 130 and 10% (v/v) heat-inactivated FCS; all from Lonza). Cells were seeded in 48-well tissue culture 131 plates (Nunc, Roskilde, Denmark) at 4×10 5 cells/500 μl/well. To each well was added either 50 μl 132 of culture medium (unstimulated), L. acidophilus NCFM or E. coli Nissle suspensions, to a final 133 concentration of 30 μg/ml. The IECs were stimulated for 2 h at 37°C in 5% CO 2 and subsequently 134 frozen in RNAlater (Qiagen). The purity of IECs was assessed by staining for the leukocyte marker 135 CD45 (phycoerythrin-labelled rat anti-mouse CD45 purchased from Abcam, Cambridge, UK) by 136 flow cytometry. IECs contained 1.3±0.4% CD45 + cells compared to 0.5±0.3% CD45 + cells in IECs 137 550 like receptor bacterial ligands in intestinal epithelial cells. Gastroenterology 126: 1054-551 1070, 2004. 552 24. Rask C, Evertsson S, Telemo E and Wold AE. A Full Flora, but not Monocolonization 553 by Escherichia coli or Lactobacilli, Supports Tolerogenic Processing of a Fed Antigen. 554 Scand J Immunol 61: 529-535, 2005. 555 25. Rimoldi M, Chieppa M, Vulcano M, Allavena P and Rescigno M. Intestinal epithelial 556 cells control dendritic cell function. Ann NY Acad Sci 66-74, 2004. 557 26. Saegusa S, Totsuka M, Kaminogawa S and Hosoi T. Candida albicans and 558 Saccharomyces cerevisiae induce interleukin-8 production from intestinal epithelial-559 like Caco-2 cells in the presence of butyric acid. FEMS Immunol Med Microbiol 41: 560
doi:10.1152/ajpgi.00428.2010 pmid:21960522 fatcat:zqxcczaimfafph7znm76ueg5lq